TW201900872A - Highly active NK cells and their utilization - Google Patents

Abstract

Provided is an NK cell having a higher cytotoxic activity. The purpose of the present invention is to provide a pharmaceutical composition for NK cell therapy that is expected to exhibit improved effects. The present invention provides an NK cell having characteristics (1) and (2) below, or a population thereof: (1) CD16 positive, CD56 high-expressive, and CD57 negative; and (2) NKG2C positive, NKG2A negative to low-expressive, and CD94 positive. The present invention also provides a pharmaceutical composition including: a population of such NK cells; and a therapeutically effective amount of an antibody.

Description

Highly active NK cells and their utilization

The invention relates to a natural killer cell (NK cell) with high cytotoxic activity and its use.

Natural killer cells (NK cells) are cytotoxic lymphocytes that function as the main factor of natural immunity. Human peripheral blood NK cells are known to be CD16-positive, to express CD56 at a low level, and to be CD57-positive (Non-Patent Documents 1 and 2).

As one of the mechanisms of cytotoxicity of NK cells, antibody-dependent cellular cytotoxicity (ADCC) is known. NK cells have Fc receptors (CD16) on their cell surfaces, and the mechanism of ADCC is that NK cells bind to antibodies bound to target cells via Fc receptors, thereby killing target cells.

NK cells are important in the rejection of tumor cells or virus-infected cells. By in vitro culture, the number of NK cells collected from the patients themselves is increased by a factor of 100 to several thousand and activated, and the NK cell therapy returned to the patients is attracting attention as a treatment method with relatively few side effects. In general, it is generally believed that about 1 × 10 ¹⁰ monocytes can be recovered by apheresis in peripheral blood of normal adults. If the composition ratio of NK cells in peripheral blood mononuclear cells is set to about 7 %, Then 7 × 10 ⁸ NK cells were obtained. On the other hand, if the patient's weight is set to 60 kg, 6 × 10 ⁶ to 4.8 × 10 ⁹ NK cells are required. Therefore, the industry is continually developing technologies for expanding NK cells obtained from donors to obtain sufficient NK cells to kill target cells. For example, Patent Document 1 proposes a method for expanding NK cells, which is characterized by including the following steps: a step of preparing a cell population containing NK cells; a step of removing T cells from a cell population containing NK cells; and using only 2500 IU IL-2 to 2813 IU / mL IL-2 as a cytokine culture medium, a step of culturing cells remaining after removing T cells without using feeder cells. [Prior Art Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. 2013-27385 (Japanese Patent No. 5572863) [Non-Patent Document]

[Non-Patent Document 1] Lorenzo, M., Blood, 116: 3689 (2010) [Non-Patent Document 2] Arima Ma, Journal of the Japanese Society of Hematopoietic Cell Transplantation, Vol. 3, No. 1: 12 (2014)

[Problems to be solved by the invention]

According to the research by the present inventors, the primary NK cells obtained from peripheral blood are highly expressive of CD16. NK cells are used as effector cells (E), K562 cells are used as target cells (T), and the mixing ratio (E : T) In the case of 1: 1 co-culture, the cytotoxic activity is about 10-20%. It is ideal if NK cells with higher cytotoxic activity are present.

In addition, most antibody medicines take ADCC activity of NK cells as one of their action mechanisms. In order for NK cells to exhibit ADCC activity, it is necessary to bind CD16 on the surface of NK cells with an antibody capable of inducing antibody-dependent cytotoxicity. However, there have been no reports on the effective combination of antibody medicine and NK cell therapy. [Technical means to solve the problem]

[1] The present invention provides the following: An NK cell or a group thereof having the following characteristics (1) and (2): (1) CD16 positive, CD56 highly expressive, and CD57 negative. (2) NKG2C positive, NKG2A negative to low expression, and CD94 positive. [2] The population according to item 1, which includes a population of NK cells with high expression of CD16 and a population of NK cells with low expression of CD16. [3] The NK cells or a group thereof described in 1, which is highly expressive of CD16. [4] The NK cells or a group thereof described in 1, which has a low expression of CD16. [5] The NK cell or the group thereof according to any one of 1 to 4, further comprising the following characteristics (3): (3) The NK cell is an effector cell (E), and the K562 cell is When the target cells (T) are co-cultured at a mixing ratio (E: T) 1: 1, the cytotoxic activity is 50% or more. [6] The method for producing NK cells or a population thereof according to any one of 1 to 5, comprising the steps of: culturing a population of primary NK cells using a medium containing IL-2 or a serum-free medium. [7] The preparation method described in 6, wherein the population of primary NK cells is subjected to a step of removing CD3-positive cells. [8] A pharmaceutical composition comprising the NK cell group according to any one of 1 to 5 and an additive permitted as a medicine. [9] A pharmaceutical composition comprising a population of NK cells and a therapeutically effective amount of an antibody. [10] The pharmaceutical composition according to 9, wherein the population of NK cells is the population of NK cells according to any one of 1 to 5. [11] The pharmaceutical composition according to 9 or 10, wherein the anti-system is capable of inducing Antibody-Dependent-Cellular-Cytotoxicity (ADCC). [12] The pharmaceutical composition according to any one of 9 to 11, wherein at least a part of the antibody binds to NK cells.

[NK Cell, NK Cell Group] The present invention provides an NK cell or a group thereof, which has the following characteristics (1): (1) CD16 positive, CD56 highly expressive, and CD57 negative. In addition, the NK cells or their populations have the following features (2): (2) NKG2C positive, NKG2A negative to low expression, and CD94 positive. The NK cell or the group thereof may further have the following characteristics (3): (3) the NK cell is an effector cell (E), the K562 cell is a target cell (T), and the mixing ratio (E: T ) In the case of 1: 1 co-culture, the cytotoxic activity was above 50%.

NK cell lines do not express T-cell receptor (TCR), CD3 as a universal marker of T cells, and large granular lymphocytes as B-cell receptors of membrane immunoglobulins. Usually humans are CD16-positive and CD56-positive. . Whether a NK cell is a practitioner or not can be easily determined based on the expression pattern of a cell surface marker or the like. NK cells have cytotoxic activity, and the presence or degree of the cytotoxic activity can be measured by various known methods. When it is referred to as NK cells, it includes various NK cells such as peripheral blood NK cells, primary NK cells, cultured NK cells, activated NK cells, and NK cells obtained by the present invention, unless otherwise specified.

<Cell Group> A cell group refers to a group of cells including a plurality of cells, for example, 1 × 10 ⁵ or more cells. The population of NK cells contains a population of NK cells with a purity of more than 50% (purity (%) = number of NK cells / number of all cells × 100). NK cells usually account for 10-30% of lymphocytes in peripheral blood of humans. That is, it is clear that the purity in peripheral blood is 50% or less. Therefore, it is considered that the population of NK cells does not exist naturally. The population of NK cells can be prepared in various cell densities. For example, it can be prepared at 1 × 10 ⁵ cells / mL or more.

The purity of the NK cells in the NK cell population provided by the present invention is preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more. The number of NK cells contained in the NK cell population is preferably 1 × 10 ⁶ cells or more, more preferably 5 × 10 ⁶ cells or more, and even more preferably 1 × 10 ⁷ cells or more. The number of NK cells contained in the NK cell population can be set to 1 × 10 ⁶ cells to 1 × 10 ¹⁰ cells suitable for administration to humans. The cell density in the NK cell population is 1 × 10 ⁵ cells / mL or more, preferably 2 × 10 ⁵ cells / mL or more, more preferably 5 × 10 ⁵ cells / mL or more, and further preferably It is 1 × 10 ⁶ cells / mL or more. The upper limit of the cell density may be, for example, 1 × 10 ¹⁰ cells / mL or less. The cell density in the NK cell population can be set to 1 × 10 ⁶ to 1 × 10 ⁸ cells / mL suitable for culture or cryopreservation, and 1 × 10 ⁵ suitable for administration to humans. ~ 1 × 10 ⁹ cells / mL.

<Feature (1)> The NK cells or a population thereof provided by the present invention has a high expression of CD16 or a low expression of CD16. In addition, the population of NK cells provided by the present invention may include both a population of NK cells with high expression of CD16 and a population of NK cells with low expression of CD16. When the cell population containing the two is analyzed for CD16 by flow cytometry, it appears bimodal. In addition, the NK cells or populations thereof provided by the present invention are highly expressive of CD56. CD56 is known as an antigen useful as a marker for NK cells. Furthermore, the expression of CD57 was not observed in the NK cells or the population thereof provided by the present invention. CD57 is known as a marker for the fifth stage (activated or mature).

Regarding CD16 and other markers, there are cases where + is positive and-is negative. For example, expressed as CD16-positive CD16 ^+, CD16 negative expressed as CD16 ^- of the situation. Positives include situations with high performance and situations with low performance. High performance may be expressed as high and bright. For example, CD16 high performance is expressed as CD16 ^high and CD16 ^bright . Low performance may be expressed as low or dim. For example, CD16 low performance is expressed as CD16 ^low and CD16 ^dim .

Positive, negative, high expression, and low expression can be judged based on the graph obtained by flow cytometry. The positions appearing in the chart may change depending on the voltage setting, sensitivity setting, antibody selection, staining conditions, use of pigment, etc. in the chart. Practitioners can use the obtained chart to identify the cell group that is identified as a group. Separate the lines appropriately.

In the determination of whether the target marker is positive or negative, the case where an isotype control antibody is used can be used as the negative control. Isotype control antibodies that do not react with specific antigens. In general, in experiments using antibodies, background may be generated due to non-specific binding to proteins other than the target or binding to Fc receptors on the cell surface. By comparing with a system using an antibody that becomes a negative control, it is clear that the response of the primary antibody to the target antigen is specific. In addition, the influence of the background can be eliminated, and the strength of the signal can be correctly interpreted.

The degree of expression of the target marker (either low expression or high expression) can be judged by comparing with the results of control cells measured under the same conditions. Examples of the control cells are NK cells which have not been substantially cultured and obtained from peripheral blood, such as the primary NK described in the item of the example of the specification of this case.

For example, the expression level of CD16 in a NK cell population can be measured by flow cytometry, and the CD16 expression in the cell population can be compared with the NK cell population obtained from peripheral blood without substantial culture (control; known as CD16 expression amount in CD16) was compared, and when the same performance situation as that of the control was observed, it was judged as high expression, and when the expression was lower than that of the control cells, it was judged as low expression.

In addition, regarding CD56, the NK cells (stage 4) are usually young (obtained by using stem cells and passed through the stages of pro-NK, pre-NK, and immature-NK) NK cells) are CD56 ^bright , and NK cells in the fifth stage (if the transcription factor MCM4 is activated in the NK cells in the fourth stage, the NK cells differentiate into the fifth stage) are understood as CD56 ^dim . Therefore, the degree of expression of CD56 in a group of NK cells can be judged by comparison with at least one of the group of NK cells in the fourth stage and the group of NK cells in the fifth stage.

The characteristics of the NK cells known so far and the NK cells obtained by the present invention are summarized below (see Non-Patent Documents 1 and 2).

[Table 1]

The above table is a conceptual diagram of a chart obtained when CD16 and CD56 are evaluated by flow cytometry. a to d correspond to the cell lines in the graph reported in Non-Patent Document 1 as follows. ^{a: CD56 bright, CD94 +++,} KIR - Killer cell Immunoglobulin-like Receptor, killer cell immunoglobulin-like receptor, CD16 ^-, perforin ^{(perforin) + -, b:} CD56 dim, CD94 ++, KIR - , CD16 ^+- , perforin ⁺ , c: CD56 ^dim , CD94 ⁺ , KIR ^+- , CD16 ⁺ , perforin ⁺⁺ , d: CD56 ^dim , CD94 ^+- , KIR ⁺ , CD16 ⁺⁺ , CD57 ⁺ , perforin ⁺⁺⁺

Primary NK cells obtained from peripheral blood are highly expressive of CD16. In addition, it is known that NK cells that are usually highly CD16-expressing are CD56 ^dim / CD57 ⁺ . CD16 ^high / CD56 ^high / CD57 ^- NK cells have not been reported in the industry so far.

<Feature (2)> Furthermore, the NK cells provided by the present invention are NKG2C positive, NKG2A negative to low expressive, and CD94 positive. NKG2A negative to low expression, CD94 performance can be observed in most NK cells. CD94 and one of the molecules of the NKG2 family are disulfide-bonded to form a receptor for MHC (Major Histocompatibility Complex) type I molecules. It is known that NKG2C is mainly expressed in NK cells and is involved in the activation of NK cells. The CD94 / NKG2C heterodimer system functions as an activation receptor. CD94 / NKG2A heterodimer is an inhibitory receptor for MHC type I. NKG2A negative to low expression includes the case of NKG2A negative, the case of extremely weak NKG2A expression, and the case of NKG2A low expression.

According to research by the inventors, the NK cells or their populations provided by the present invention are CD94 ⁺⁺ , KIR ⁺ , and NK cells as compared with the NK cells reported in Non-Patent Document 1 (see a to d above), and Perforin ⁺⁺⁺ .

The NK cells or populations thereof provided by the present invention can be distinguished from the existing NK cells or populations by the characteristics of (2), or they can be used instead of the characteristics of (2) by the following (2-1) Distinguish from existing NK cells or their populations. (2-1) KIR (s) (Killer cell Immunoglobulin-like Receptor (s)) was positive.

The KIR (s) -positive means at least one member selected from the group consisting of KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1.

The NK cells or their populations provided by the present invention can be distinguished from the existing NK cells or their populations by the features of (2), or they can be used instead of the features of (2) by the following (2-2) Distinguish from existing NK cells or their populations. (2-2) Any one selected from the group consisting of NKp44 positive, NKp30 high expression, and NKp46 high expression is satisfied, preferably any two, more preferably all.

NKp44 is usually negative in primary NK cells. NKp30 is a NK-specific target receptor that triggers non-limiting cytotoxicity of MHC. It is one of NKp46, NKp44 and the natural cytotoxicity receptor (NCR, Natural Cytotoxicity Receptor) identified so far. It is known that NKp30 behaves in parallel with NKp46, and NK cells that are NKp46 ^bright are NKp30 ^bright . It is also known that NKp30 ^bright line is associated with a high cytotoxic activity. Regarding the characteristics imparted by the NK cells or their populations by (2-2) provided by the present invention, reference can be made to Example 1 and FIG. 1 of the specification of this case.

For the expression pattern of surface markers at each stage of differentiation of NK cells, refer to the following documents. Non-Patent Document 3: Luetke-Eversloh M., M. Killig, C. Romagnani. 2013. Signatures of human NK cell development and terminal differentiation. Front. Immunol. 4: 499. PubMedGoogle Scholar

<Feature (3)> The NK cells or the group thereof provided by the present invention can exhibit high cytotoxic activity. The so-called cytotoxic activity refers to the lytic ability of a target cell (effector cell, E) to a target cell (T), unless otherwise specified. The cytotoxic activity can be expressed as a percentage (%) of target cells that die due to effector cells, and can be obtained by the following formula.

(Cell death in the case of co-culture with effector cells-natural cell death (negative control)) / (maximum cell death (positive control)-natural cell death (negative control)) x 100

In the measurement of cytotoxic activity, usually according to the degree of cytotoxic activity of effector cells, the mixing ratio of effector cells to target cells (E: T), and the time of co-cultivation of effector cells and target cells can be determined according to the cell used. Kind or intensity of activity is appropriate. When NK cells are used as effector cells, the target cells include K562 cells, acute epiphyseal leukemia cells, and chronic epiphyseal leukemia cells, but the present invention is not limited to these. Effector cells and target cells, live cells and dead cells can be distinguished and quantified by reagents such as antibodies labeled with radioactive substances, fluorescent pigments, and the like. The cytotoxic activity when NK cells are set as effector cells can be measured, for example, under the following conditions: K562 cells are set as target cells, and E: T = 1: 0.05 to 10, preferably 1: 0.1 to 5 and the culture time is 0.5 to 18 hours, preferably 1 to 12 hours.

The target cell is a K562 cell from the NK cell or its lineage provided by the present invention, and the cell is cytotoxic when mixed with E: T = 1: 1 and co-cultured for 1 to 3 hours, or 2 hours if further specified. The activity is 50% or more, preferably 60% or more, and more preferably 70% or more.

The cytotoxic activity of the primary NK cells obtained from peripheral blood was low. To date, the industry has not reported NK cells that are CD16 ^high / CD56 ^high / CD ^TM 57 ^- and have high cytotoxic activity.

[Method for preparing NK cells or a population thereof] The present invention provides a method for preparing the above-mentioned NK cells or a population thereof, comprising the steps of: culturing a population of primary NK cells using a medium containing IL-2.

<Primary NK cells> In the preparation method of the present invention, the population of primary NK cells can be obtained by a step of separating monocytes from blood cells collected from a subject. Blood cells may be collected from peripheral blood, umbilical cord blood, callus, and / or lymph nodes. Blood cells may be collected from peripheral blood by hematocrit.

In the preparation method of the present invention, the population of primary NK cells may be prepared by using a cell selected from the group consisting of cells derived from embryonic stem cells, adult stem cells, and artificial pluripotent stem (iPS) cells. Hematopoietic stem cells derived from any of stem cells, hematopoietic stem cells derived from umbilical cord blood, hematopoietic stem cells derived from peripheral blood, hematopoietic stem cells derived from epiphyseal blood, cord blood mononuclear cells, peripheral blood mononuclear cells At least 1 type of cell. The subject who is the donor of the primary NK cell population may be derived from the patient who is the recipient, or a close relative of the patient, or a person who is not related to the patient. Subjects are healthy and sick. NK cells may have donors whose major histocompatibility antigen (MHC) derived from the recipient is inconsistent with the Killer Immunoglobulin-like Receptor (KIR).

In the preparation method of the present invention, T cells can be removed from the population of primary NK cells. The removal of T cells is sometimes achieved by a step of removing CD3-positive cells.

The method for preparing the NK cells of the present invention may include a step of removing precursor cells from a hematopoietic population of primary NK cells. The step of removing hematopoietic precursor cells from a population of NK cells may be achieved by a step of removing CD34 positive cells.

The population of primary NK cells can be prepared using various sequences known to practitioners. For example, when recovering monocytes from blood such as umbilical cord blood and peripheral blood, a specific gravity centrifugation method can be used. NK cells can be collected using immunomagnetic beads. Furthermore, the population of primary NK cells can be immunofluorescently stained with specific antibodies to cell surface markers, and isolated using FACS (Fluorescence activated cell sorter) or flow cytometry. Identification. In addition, the population of primary NK cells can be separated and removed by using immunobeads including Dynabeads (trademark) manufactured by Dynal Inc. sold by Invitrogen or CliniMACS (trademark) produced by Miltenyi Biotec, but not limited to these And / or CD34 cells. In addition, there is a case where the T cells and / or hematopoietic precursor cells are specifically bound to selectively poison or kill T cells and / or hematopoietic precursor cells. Furthermore, the step of removing T cells may be a step of removing other cell types, such as hematopoietic precursor cells, B cells, and / or Natural Killer T (NKT) cells, together with T cells. The step of removing hematopoietic precursor cells may be a step of removing other cell types, such as T cells, B cells, and / or NKT cells, together with hematopoietic precursor cells.

<Media> Cell culture medium for culturing primary NK cell populations includes KBM501 medium (Kohjin Bio Co., Ltd .; contains IL-2 at 1,750 JRU / ml; for primary culture of human NK cells (Primary Culture)), CellGro SCGM Medium (CellGenix, Iwai Chemical Co., Ltd.), X-VIVO15 medium (Lonza, TAKARA BIO Co., Ltd.), COSMEDIUM 008 (Cosmo Bio; IL-2 containing 1,750 JRU / ml; for primary culture of human NK cells), CTS AIM V Medium Gibco ^TM CTS ^TM AIM V ^TM Medium (Thermo Fisher Scientific; a serum-free medium of known composition for the proliferation and manipulation of T cells and dendritic cells), CTS OpTmizer T cell expansion base medium (CTS OpTmizer T Cell Expansion Basal Medium) (Thermo Fisher Scientific; for the growth and proliferation of human T lymphocytes), IMDM (Iscove's Modified Dulbecco's Medium), MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium (Duber can modify Eagle's medium), RPMI-1640, etc., but is not limited to these.

There is a case where interleukin-2 (IL-2) is added to the culture medium at a concentration that can achieve the purpose of the present invention. The IL-2 concentration may range from 2500 IU / mL to 2813 IU / mL. IL-2 preferably has a human amino acid sequence, and is preferably produced by recombinant DNA (Deoxyribonucleic Acid) technology in terms of safety. The concentration of IL-2 may be expressed in domestic standard units (JRU) and international units (IU). Since 1 IU is about 0.622 JRU, the existing medium at 1750 JRU / mL is equivalent to about 2813 IU / mL.

In some cases, the subject's autologous serum, human AB serum that can be obtained from BioWhittaker and other companies, or blood donated human serum albumin that can be obtained from the Japanese Red Cross may be added to the culture medium. Autologous serum and human AB serum are preferably added at a concentration of 1 to 10%, and blood donated human serum albumin is preferably added at a concentration of 1 to 10%.

In some cases, appropriate proteins, cytokines, antibodies, compounds, and other components may be contained in the culture medium under conditions that do not impair the expansion effect of NK cells. The cytokines are interleukin 3 (IL-3), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin-15 (IL-15), interleukin-21 ( IL-21), stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (Flt3L). IL-3, IL-7, IL-12, IL-15, IL-21, SCF and Flt3L preferably have human amino acid sequences, and are preferably produced by recombinant DNA technology in terms of safety.

The medium is preferably a serum-free medium. The serum-free medium preferably contains serum albumin, transferrin, and insulin. Serum-free medium for culturing lymphocytes has been developed and is commercially available, and these can be utilized in the present invention. One of the preferred examples of the serum-free medium is a commercially available CTS Immune Cell SR (Thermo Fisher Scientific) added to the basal medium as a component that supports the proliferation of human T cells.

The replacement of the culture medium can obtain the required number of NK cells as a condition, and is performed at any time after the start of the culture, preferably every 3 to 5 days.

The culture vessel used in the culture includes commercially available culture dishes, flasks, culture plates, and multiwell plates, but is not limited thereto. The culture condition is a condition that does not impair the expansion effect of the NK cells, and is not particularly limited. Usually, the culture conditions are 37 ° C, 5% CO ₂ and a saturated water vapor atmosphere. The longer the culture period using the medium, the more NK cells can be obtained, which is advantageous. The conditions for the expansion of NK cells to a desired number of cells during the culture are not particularly limited.

The specific NK cell population obtained by culture may include NK cell precursors, T cells, NKT cells, hematopoietic precursor cells, etc. in addition to the target NK cells. After culturing, the target NK cells or their populations may be selected using, for example, specific gravity centrifugation, immunomagnetic beads, FACS, flow cytometry, or the like. For example, there are anti-CD3 antibodies, anti-CD16 antibodies, anti-CD34 antibodies, anti-CD56 antibodies, anti-CD69 antibodies, anti-CD94 antibodies, anti-CD107a antibodies, anti-KIR3DL1 antibodies, anti-KIR3DL2 antibodies, anti-KIR2DL3 antibodies, anti-KIR2DL1 antibodies, anti-KIR2DS1 antibodies , Anti-KIR2DL5 antibody, anti-NKp46 antibody, anti-NKp30 antibody, anti-NKG2D antibody, etc., to selectively isolate the target NK cell or its population. The antibody may be a single antibody, a multiple antibody, or the like. Selection of target NK cells or populations is performed by selectively removing T cells, NKT cells, hematopoietic precursor cells, and other cells.

[Usage of NK cells or populations thereof] The present invention provides a pharmaceutical composition comprising a population of NK cells and an additive permitted as a medicine. The present invention also provides a pharmaceutical composition comprising a population of NK cells and a therapeutically effective amount of an antibody. Examples of the additives allowed as medicines include isotonic agents, pH adjusters, buffers, stabilizers, cryoprotectants, antibiotics, and the like. Specific examples include water, ethanol, sodium chloride, glucose, albumin, and the like. The population of NK cells contained in the pharmaceutical composition of the present invention is preferably the population of NK cells provided by the present invention. In addition, the population of NK cells contained in the pharmaceutical composition of the present invention may be highly expressive of CD16, and in any case, it is more preferably a population of NK cells with high cytotoxicity.

The antibody used in the pharmaceutical composition of the present invention is preferably one that can be used as an antibody drug. In addition, those capable of inducing Antibody-Dependent-Cellular-Cytotoxicity (ADCC) are preferred. The antibody used in the pharmaceutical composition of the present invention may be any of a mouse antibody, a chimeric antibody, a humanized antibody, and a human antibody, and is preferably a humanized antibody or a human antibody. The antibody used may be modified or produced by Potelligent technology (removing fucose from the Fc region of IgG (Immunoglobulin G)).

Specific examples of the antibody that can be used as an antibody pharmaceutical and can be used in the pharmaceutical composition of the present invention include ibrumumoma tiuxetan, iodine 131, catumaxomab, Bonatuzumab (blinatumomab), Morozumab-CD3 (muromonab-CD3), abciximab, rituximab, basiliximab, infliximab Anti (infliximab), cetuximab, bentuximab, siltuximab, dinutuximab, obitoxaximab, Daclizumab, palivizumab, trastuzumab, gemtuzumab, alemtuzumab, omalizumab ), Efalizumab, bevacizumab, natalizumab, tocilizumab, ranibizumab, eculizumab MAb (eculizumab), certolizumabpegol, mogamulizumab, pertuzumab, song Trastuzumab, atosuzumab, obututumumab, vedolizumab, pembrolizumab, idarucizumab, mepolizumab ( mepolizumab), elotuzumab, daratumumab, ixekizumab, reslizumab, adalimumab, panitumumab Anti (panitumumab), golimumab, ustekinumab, canakinumab, ofatumumab, denosumab, ipilimumab Antibody (ipilimumab), belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, Ivocurumab Evolocumab, alirocumab, and necitumumab.

The antibody used in the pharmaceutical composition of the present invention is preferably one having a higher affinity for CD16. In the pharmaceutical composition of the present invention, it is preferable that at least a part of the antibody binds to NK cells. According to research by the present inventors, in the case of using mogazumab produced using Potelligent technology as an antibody, a system in which antibodies are present in co-culture of NK cells and target cells, and NK cells and antibodies are mixed in advance, And in a system in which co-culture is performed after washing NK cells (removing antibodies that are not bound to the cells) before co-culturing with target cells, a higher cytotoxic activity can be observed for the latter. On the other hand, when dastuximab was used under the same conditions, the effect caused by mixing NK cells and antibodies in advance was not observed. This situation suggests that as long as the antibody has a high affinity for CD16, the antibody can bind to NK cells by mixing with the NK cells in advance, thereby exhibiting a higher ADCC activity. Furthermore, if the conditions are studied, it is considered that it is possible to obtain the same effect even with dastuximab.

The pharmaceutical composition of the present invention may be prepared during use. For example, the pharmaceutical composition of the present invention can be prepared by maintaining the NK cell population and the antibody in another container, and mixing the NK cell population and the antibody immediately before administration to a subject several hours before.

The pharmaceutical composition of the present invention can also be prepared by a step of removing an antibody that is not bound to NK cells after mixing the NK cell population with the antibody. That is, one of the preferable aspects of the pharmaceutical composition of the present invention is that although the NK cells and the antibodies are included, the antibodies are bound to the NK cells and substantially free of the antibodies that are not bound to the NK cells.

It is thought that most of the antibody drugs exhibited antitumor effects due to ADCC activity after intravenous administration. On the other hand, when exhibiting ADCC activity, in addition to NK, monocytes / macrophages or neutrophils are also mobilized. In addition, effectors other than NK cells show ADCC activity without distinguishing between normal cells and cancer cells, which is thought to cause side effects. In the present invention, the NK cells are provided with the antibody in advance by mixing the NK cells and the antibody before administration. Therefore, it is thought that the effector is limited to NK cells, which can reduce the amount of antibody administered, and is also very effective in reducing side effects.

The pharmaceutical composition of the present invention may have an HLA (Human Leukocyte Antigen, human leukocyte antigen) genotype different from the NK cells prepared by the preparation method of the present invention, and may be administered to a patient.

A pharmaceutical composition is typically a form in which NK cells are suspended in a solution. The solution for suspending NK cells is usually, for example, a cryoprotective solution containing DMSO (dimethylsulfoxide, dimethylsulfoxide), physiological saline, phosphate buffered saline (PBS), culture medium, serum, and the like. The solution may contain a pharmaceutically acceptable carrier as a pharmaceutical or quasi-drug.

The pharmaceutical composition of the present invention is useful for treating infectious diseases or cancer. In addition, the pharmaceutical composition of the present invention can be applied to the treatment and / or prevention of various diseases that are sensitive to NK cells. Diseases include, but are not limited to, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, colorectal cancer, kidney cancer, bladder cancer, leukemia, or infections caused by viruses, bacteria, and the like. The cell therapy of the present invention may be implemented alone or in combination with surgical therapy, chemotherapy, radiation therapy, antibody medicine, and the like. In the cell therapy using the pharmaceutical composition of the present invention, for example, NK cells may be administered to a vein, an artery, subcutaneously, or intraperitoneally.

The manufacturing of the pharmaceutical composition of the present invention is preferably in accordance with the conditions (GMP (good manufacturing practice)) and manufacturing management and quality management of products such as regenerative medicine in compliance with the manufacturing management and quality management rules for pharmaceuticals and quasi-drugs. (GCTP (Good Gene, Cellular, and Tissue-based Products Manufacturing Practice)).

In the present invention, the collection of whole blood of umbilical cord blood and peripheral blood, the preparation of autologous serum, the preparation of monocytes derived from whole blood, the measurement of the number of cells before and after the culture of the monocytes, and the single list before and after the culture NK cells, T cells, hematopoietic precursor cells, and other types of cell types in nuclear cells can be measured, the expansion ratio of NK cells can be calculated, and statistical analysis of measurement errors or significance can be used by any practitioners Method.

The embodiments of the present invention described below are merely examples and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the scope of patent applications. Changes to the present invention, such as additions, deletions, and replacements of the constituent elements of the present invention can be made without departing from the gist of the present invention. [Example]

[Example 1] Blood was collected from healthy human volunteers, and the obtained peripheral blood mononuclear cells were fractionated using polysucrose (GE Healthcare, 17144002). CD3 magnetic beads ^{※ 1} were added to the obtained peripheral blood mononuclear cells and suspended. After incubation at 4 ° C for 15 minutes, the separation buffer 1 ^{※ 2 was} added to fully suspend and 300 × g for 10 minutes. Centrifuge. The supernatant was removed, suspended in 1 mL of separation buffer, and added to a LD column (Miltenyi Biotec, 130-042-901) wetted with 2 mL of separation buffer previously added, and recovered from the LD column Of the eluate. Furthermore, 1 mL of the separation buffer was added to the LD column, and the eluate was recovered. Thereafter, the column was washed twice, and the number of cells in the recovered solution was counted to calculate the total number of cells. After performing centrifugation at 500 × g for 5 minutes, the supernatant was removed, and then suspended in NK medium I ^{* 3} so as to be 5 × 10 ⁵ cells / mL. A part of the cells was collected for flow cytometry measurement (here, the recovered cells are referred to as "Primary NK"), and the remaining cells were cultured. The culture system was performed using a 6-well plate (Thermo Fisher Scientific, 140675) or a T-75 flask (Thermo Fisher Scientific, 156499) in a CO ₂ incubator (37 ° C, 5% CO ₂ ). On the 5th and 9th days of culture, one part of the culture solution was taken to count the number of cells, and NK medium I was added so that the cell density became 5 × 10 ⁵ cells / mL. Cell recovery was performed on the 14th day (herein, the recovered cells are referred to as "Day 14"), and a part of the cells was used to measure the cell surface antigen using a flow cytometer. The remaining cell lines were suspended in STEM-CELLBANKER GMP grade (TAKARA BIO, CB045) and stored at -80 ° C.

The recovered cells were stained with the following antibodies: BV421-labeled anti-human CD56 antibody (Biolegend, 318328), PerCP (Peridinin Chlorophyll Protein) /C5.5 at a concentration of 1 μg / mL Labeled anti-human CD3 antibody (Biolegend, 300430), APC (Antigen Presenting Cell) labeled anti-human NKp30 antibody (Biolegend, 325209), PE (phycoerythrin, phycoerythrin) labeled anti-human NKp44 antibody (Biolegend, 325107) FITC (fluorescein isothiocyanate, fluorescent isothiocyanate) labeled anti-human NKp46 antibody (Biolegend, 331921), PE-Cy7 labeled anti-human NKG2D antibody (Biolegend, 320811), PE-Cy7 labeled anti-human CD16 antibody (Biolegend, 302015), APC-labeled anti-human CD69 antibody (Biolegend, 310910), FITC-labeled anti-human CD94 antibody (Biolegend, 305504), FITC-labeled anti-human CD14 antibody (Biolegend, 325604), PE-labeled anti-human NKG2C antibody (R & D, FAB138P) APC-labeled anti-human NKG2A antibody (Milteni Biotec, 130-098-809), PerCP Cy5.5-labeled anti-human CD94 antibody (Biolegend, 305514), APC-labeled anti-human CD57 antibody (Biol egend, 359610) After dyeing at 4 ° C for 30 minutes, centrifuge (500 × g, 5 minutes, 4 ° C), remove the supernatant, and suspend it in PBS (Wako Pure Chemical Industries), then use flow cytometry. The measurement was performed with a cytometer (BD LSRFortessa, BD Bioscience), and analysis was performed using FlowJo software (TreeStar).

※ 1: CliniMACS CD3, Miltenyi Biotec, catalog number 130-017-601 (5 μs per 1 × 10 ⁷ cells) ※ 2: Contains 0.5% human AB type serum (Cosmo Bio, 12181301 at 30 ° C for 30 Minute inactivation treatment), 2 mM EDTA (Ethylenediamine Tetraacetic Acid) (Thermo Fisher Scientific, 15575-020) in PBS ※ 3: 5% human AB type serum (Cosmo Bio (12181301 inactivated at 56 ° C for 30 minutes) COSMEDIUM 008 (Cosmo Bio, COS-008)

The results are shown in FIG. 1. On the 14th day, NK cells exhibited a state of so-called highly activated NK cells that strongly expressed activated receptors such as NKp46, NKp30, and NKG2D. On the other hand, the performance of CD16 is bimodal.

[Example 2] Cells were prepared by the method described in Example 1. A part of the cells at the beginning of the culture and the cells at the end of the culture (day 14 of the culture) were recovered, and the cell surface antigen was measured using a flow cytometer. About 1 × 10 ⁵ cells were recovered at a concentration of 1 μg / mL to BV421-labeled anti-human CD56 antibody, PerCP / C5.5-labeled anti-human CD3 antibody, PE-Cy7-labeled anti-human CD16 antibody, and APC-labeled anti-human CD69 antibody and FITC-labeled anti-human CD14 antibody were stained at 4 ° C for 30 minutes, then centrifuged (500 × g, 5 minutes, 4 ° C), the supernatant was removed, and the suspension was suspended in an appropriate amount of PBS. Flow cytometry (BD LSRFortessa, BD Bioscience) was used for measurement, and analysis was performed using FlowJo software. CD3-negative CD56-positive cells were designated as NK cells, and CD16 and CD56 were used to expand and analyze the expression of CD16 on NK cells.

The results are shown in FIGS. 2A to 2C. NK cells on day 14 consisted of CD56 ^high / CD16 ^high NK cells ((i) population) and CD56 ^high / CD16 ^low NK cells ((ii) population) (Figure 2A) and were not observed and were generally considered to be stage 5 The expression of CD57 (activated or mature) marker (Figure 2B). It was found that the NK cells on the 14th day were further characterized by NKG2C positive, NKG2A negative to low expression, and CD94 positive (Fig. 2C).

[Example 3] A part of the method described in Example 1 was changed to prepare cells. Specifically, culture was performed using separation buffer 2 ^{* 4} and NK medium II ^{* 5} . A part of the cells at the beginning of the culture and the cells at the end of the culture (the 14th day of the culture) were recovered and measured and analyzed by the method described in Example 2.

※ 4: 0.5% CTS ^TM Immune Cell SR (Thermo Fisher Scientific, A25961-01, hereinafter referred to as CTS), 2 mM EDTA in PBS ※ 5: COSMEDIUM 008 medium (Cosmo with 5% CTS) Bio, COS-008)

The results are shown in FIG. 3. The serum-free medium supplemented with CTS can increase the ratio of (i) population to the entire NK cells.

[Example 4] ≫Preparation of K562 (target cells) ≫ Using 10% FBS (Fetal Bovine Serum, fetal bovine serum) (Nichirei Bioscience, 171012-500ML) and 100 units of penicillin, 100 μg / mL of streptomycin (Nacalai Tesque, 26253-84) RPMI1640 medium (Wako Pure Chemical Industries, Ltd., 189-02025) (hereinafter, referred to as 10% FBS / RPMI1640) K562 cells (human chronic epiphysis) were prepared at a concentration of 1 × 10 ⁶ cells / ml Leukemia cell line). DiOC18 (Sigma, D4292-20MG) was added to the prepared K562 cells so that the final concentration became 30 μM, and reacted at 37 ° C. for 10 minutes. After centrifugation (400 × g, 5 minutes, room temperature), the supernatant was removed, and 10% FBS / RPMI1640 was added and suspended. This washing step was repeated three times, and finally, 10% FBS / RPMI1640 was used to prepare 2 × 10 ⁶ cells / mL.

≫Preparation of NK cells (effect cells) ≫ After recovering and washing the cells obtained by the method described in Example 1 from healthy volunteers, the cells were suspended with 10% FBS / RPMI1640, and the same medium was used. Prepared at a concentration of 1 × 10 ⁶ cells / ml. The prepared cell suspension was stained with PE-Cy7 labeled anti-human CD16 antibody (1 μg / mL) at 4 ° C for 30 minutes, then centrifuged (500 × g, 5 minutes, 4 ° C), and the supernatant was removed. liquid. After suspending the cells with 10% FBS / RPMI1640, the same culture medium was used to prepare a concentration of 2 × 10 ⁷ cells / ml, and the cell surface antigen was measured using a flow cytometer (Cell Sorter SH800, SONY) and confirmed. The performance of CD16. The same cell sorter was used to sort CD16 ^high NK cells and CD16 ^dim NK cells, and recovered them into 15 ml conical tubes filled with 7 ml of 10% FBS / RPMI1640. After recovery, centrifugation (500 × g, 5 minutes, 4 ° C) was performed. After removing the supernatant, the suspension was suspended with 10% FBS / RPMI1640, and the same medium was used to prepare 2 × 10 ⁶ cells / ml. concentration.

≫Determination of cytotoxicity≫ 10% formalin was also used to separate unsorted NK cells and K562 cells, CD16 ^high NK cells and K562 cells, CD16 ^dim NK cells and K562 cells, only each Six groups of NK cells, a group of K562 cells serving only as a negative control, and a group of K562 cells serving as a positive control were fixed. NK cells and K562 cell lines were added to a 96-well plate so that the cell ratio became 1: 1, mixed, and co-cultured at 37 ° C for 2 hours. At this time, K562 cells were first added to the culture plate, then 200 μg / ml of APC-labeled anti-human CD107a antibody ^{※ 6} (Biolegend, 328620) was added so that the final concentration became 1 μg / ml, and each NK cell was finally added. After incubation, centrifuge (500 × g, 5 minutes, 4 ° C), remove the supernatant, add 7-AAD solution (Beckman Coulter, A07704) diluted with PBS, and suspend it in the chamber. Incubate at room temperature for 10 minutes. Measurement was performed using a flow cytometer (BD LSR Fortessa, BD Bioscience), and analysis was performed using FlowJo software.

The cytotoxic activity was calculated by the following formula. The calculation is performed in the same manner in the following examples. (Cell death of effector cells and target cells in the case of culture-natural cell death (negative control)) / (maximum cell death (positive control)-natural cell death (negative control)) x 100

* 6: CD107a is present on the granules in NK cells and transferred to the surface of the cell membrane when degranulation (perforin, granzyme release). Therefore, positive CD107a indicates that NK attacked the subject.

≪Results The results are shown in FIG. 4. The cytotoxic activity of group (i) and group (ii) both exceeded 50%.

[Example 5] ≫Preparation of IMR32 cells (target cells) ≫ 10% FBS / RPMI1640 was used to prepare IMR32 cells (human MYCN-expanded neuroblastoma cell line) to a concentration of 1 × 10 ⁶ cells / ml. DiOC18 (Sigma, D4292-20MG) was added to the prepared K562 cells so that the final concentration became 30 μM, and reacted at 37 ° C. for 10 minutes. After centrifugation (400 × g, 5 minutes, room temperature), the supernatant was removed, and 10% FBS / RPMI1640 was added and suspended. This washing step was repeated 3 times, and finally 10% FBS / RPMI1640 was used to prepare 2 × 10 ⁶ cells / mL.

≫Preparation of NK cells (effect cells) ≫ After recovering and washing the cells obtained by the method described in Example 1 from healthy volunteers, the cells were suspended with 10% FBS / RPMI1640, and the same medium was used. Prepared at a concentration of 1 × 10 ⁶ cells / ml. The preparation was stained with PE-Cy7-labeled anti-human CD16 antibody (1 μg / mL) at 4 ° C for 30 minutes, and then centrifuged (500 g, 5 minutes, 4 ° C), and the supernatant was removed. After suspending the cells with 10% FBS / RPMI1640, the cells were prepared at a concentration of 2 × 10 ⁷ cells / ml in the same medium, and the cell surface antigen was measured using a flow cytometer (Cell Sorter SH800, SONY) to confirm The performance of CD16. Use the same cell sorter to sort CD16 ^high NK cells and CD16 ^dim NK cells and recover them into 15 ml conical tubes filled with 7 ml of 10% FBS / RPMI1640. After recovery, centrifugation (500 g, 5 minutes, 4 ° C) was performed. After removing the supernatant, the suspension was suspended with 10% FBS / RPMI1640, and the same culture medium was used to prepare a concentration of 2 × 10 ⁶ cells / ml. .

≫Determination of cytotoxicity≫ 10% formalin was also used to separate unsorted NK cells and K562 cells, CD16 ^high NK cells and K562 cells, CD16 ^dim NK cells and K562 cells, only each Six groups of NK cells, a group of K562 cells serving only as a negative control, and a group of K562 cells serving as a positive control were fixed. NK cells and K562 cell lines were added to a 96-well plate so that the cell ratio became 1: 1, mixed, and co-cultured at 37 ° C for 2 hours.

At this time, first add IMR32 cells to the culture plate, and then add 1 μl each of dastuximab (trade name Unituxin, United Therapeutics) prepared with PBS (100 μg / ml) (dastuximab addition group). Then, 200 μg / ml of APC-labeled anti-human CD107a antibody was added such that the final concentration became 1 μg / ml, and each NK cell was finally added. After co-culture, centrifuge (500 g, 5 minutes, 4 ° C), remove the supernatant, add 7-AAD solution (Beckman Coulter, A07704) diluted with PBS, and suspend it in the chamber. Incubate at room temperature for 10 minutes. Measurement was performed using a flow cytometer (BD LSR Fortessa, BD Bioscience), and analysis was performed using FlowJo software.

≪Results The results are shown in FIG. 5. Higher cytotoxic activity was observed for the dastuximab addition population.

[Example 6] ≫Preparation of cells≫ The NK cells obtained by the method of Example 1 were suspended in mogazumab (POTELIGEO (registered trademark)) at a concentration of 4 μg / mL (POTELIGEO (registered trademark) 20 mg, Kyowa Hakko Kirin) was cultured at 37 ° C for 20 minutes. After centrifugation (500 × g, 5 minutes, room temperature), remove the supernatant, add 10% FBS / RPMI1640 and suspend it, and then centrifuge (500 × g, 5 minutes, room temperature), remove the supernatant The supernatant was prepared at 10% FBS / RPMI1640 to 2 × 10 ⁶ cells / mL, and was prepared into NK + mogazumab (premix).

To Hut 78 cells (human T lymphoma cell line), DiOC18 (Sigma, D4292-20MG) was added so that the final concentration became 1 μM, and the reaction was performed at 37 ° C. for 10 minutes. After centrifugation (500 × g, 5 minutes, room temperature), the supernatant was removed, and 10% FBS / RPMI1640 was added and suspended. This washing step was repeated three times, and finally, 10% FBS / RPMI1640 was used to prepare 2 × 10 ⁶ cells / mL.

≫Measurement of cytotoxic activity≫ A group of 5 (cell number ratio) for Hut 78 cells relative to NK cell 1 and 5 (cell number ratio) for Hut 78 cells relative to NK cell 1 was prepared. A group of 4 μg / mL to which mogazumab was added, a total of 3 groups of a group of 5 (cell number ratio) with Hut 78 cells relative to NK + mogazumab (premix) 1 was added. The cells were added to a 96-well plate at a cell ratio of 1: 5, mixed, and co-cultured at 37 ° C for 2 hours. A group of Hut 78 cells only as a negative control and a group of Hut 78 as a positive control were fixed with 10% formalin. After 2 hours of co-cultivation, centrifugation (500 × g, 5 minutes, room temperature) was performed, the supernatant was removed, and a 7-AAD solution (Beckman Coulter, A07704 diluted 6-fold with PBS was added at 60 μL / well). ), Suspended, and incubated at room temperature for 10 minutes. Measurement was performed using a flow cytometer (BD LSRFortessa, BD Bioscience), and analysis was performed using FlowJo software.

制备 Preparation of IMR32 cells and determination of cytotoxic activity≫ It was performed according to the experiment in Hut 78 described above. The difference is that the cell line used is IMR32, the antibody used is dastuximab, and the cell ratio of NK cells to tumor cells is 1:10.

≪Results The results are shown in FIG. 6. Regarding mogazumab, the highest cytotoxic activity was observed for the NK + mogazumab (premix) population. Most antibody medicines use antibody-dependent cytotoxic (ADCC) activity of NK cells as one of their mechanisms of action, suggesting the effectiveness of mixing NK cells with antibody medicines in advance.

[Example 7] NK cells obtained by the method described in Example 1 were frozen using STEM-CELLBANKER GMP grade (TAKARA BIO, CB045), and then thawed and NK medium II was used at 37 ° C in CO ₂ Incubate overnight in the incubator. Cells were detached with PBS containing 1 mM EDTA (Thermo Fisher Scientific, 15575-020), 1 × 10 ⁵ cells were dispensed into round bottom 96-well plates (IWAKI), and centrifuged (500 × g, 5 minutes, chamber) After removing the supernatant, add 100 μL of mogazumab solution (final concentration: 0 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, 1,000 μg / mL). It was suspended and incubated at room temperature for 1 hour. After centrifugation (500 × g, 5 minutes, room temperature), the supernatant was removed, and three washing operations (500 × g, 5 minutes, room temperature) were performed with 300 μL of PBS. Next, add the antibody solution for staining (BV421-labeled anti-human CD56 antibody, PerCP / C5.5-labeled anti-human CD3 antibody, PE-Cy7-labeled anti-human CD16, PE-labeled anti-human IgG Fc antibody (SouthernBiotech, 2043-09), It was suspended and reacted for 30 minutes at 4 ° C. After centrifugation (500 × g, 5 minutes, room temperature), the supernatant was removed, 100 μL of PBS was added, and the suspension was suspended using a flow cytometer (BD LSRFortessa, BD Bioscience) was measured and analyzed using FlowJo software.

The results are shown in FIG. 7. Moguzumab was confirmed to bind to NK cells. Not only did mogazumab successfully bind to group (i), but it also successfully combined mogazumab with group (ii) when the concentration of mogazumab was high.

Figure 1 shows the performance of major cell surface markers in NK cells on day 14 of culture. CD16 is bimodal. Figure 2A shows the characteristics of NK cells on the 14th day characterized by CD56 and CD16. CD56 ^high / CD16 ^high NK cells ((i) population) and CD56 ^high / CD16 ^low NK cells ((ii) population) were included on day 14. Figure 2B shows the characteristics of NK cells on day 14. The performance of CD57, which is considered to be a marker of Stage 5 (activated, or mature), is generally not observed. Figure 2C shows the characteristics of NK cells on day 14. NKG2C positive, NKG2A negative to low expression, CD94 positive. FIG. 3 shows NK cells on the 14th day obtained by culturing using serum-free and CTS supplemented medium. It is possible to increase the ratio of the (i) population to the entire NK cells. Figure 4 shows the cytotoxic activity of groups (i) and (ii). The cytotoxic activity of group (i) and group (ii) both exceeded 50%. Figure 5 shows the cytotoxic activity of groups (i) and (ii). Higher cytotoxic activity was observed for the dinutuximab addition population. Figure 6 shows the effect of antibody medicine + NK cells. Regarding Mogamulizumab, the highest cytotoxic activity was observed for the premix group (premix group). Figure 7 shows the analysis results of mogazumab and cultured NK cells. Binding of mogazumab to NK cells was confirmed. Not only did mogazumab successfully bind to group (i), but it also successfully combined mogazumab with group (ii) when the concentration of mogazumab was high.

Claims (12)

An NK cell or a group thereof having the following characteristics (1) and (2): (1) CD16 positive, CD56 highly expressive, and CD57 negative; (2) NKG2C positive, NKG2A negative to low expressiveness , And CD94 positive. The population of claim 1 includes a population of NK cells with high expression of CD16 and a population of NK cells with low expression of CD16. The NK cells or populations thereof as claimed in claim 1 are highly CD16 expressive. The NK cells or populations thereof as claimed in claim 1 have low CD16 expression. The NK cell or the group thereof according to any one of claims 1 to 4, further has the following characteristics (3): (3) the NK cell is an effector cell (E), and the K562 cell is a target cell ( T), and the cytotoxic activity in the case of co-culture at a mixing ratio (E: T) of 1: 1 is 50% or more. A method for preparing NK cells or a population thereof according to any one of claims 1 to 5, comprising the steps of: culturing a population of primary NK cells using a medium containing IL-2, or a serum-free medium. The method of claim 6, wherein the population of primary NK cells is subjected to a step of removing CD3-positive cells. A pharmaceutical composition comprising the NK cell population according to any one of claims 1 to 5 and an additive permitted as a medicine. A pharmaceutical composition comprising a population of NK cells and a therapeutically effective amount of an antibody. The pharmaceutical composition according to claim 9, wherein the population of NK cells is the population of NK cells according to any one of claims 1 to 5. The pharmaceutical composition according to claim 9 or 10, wherein the anti-system is capable of inducing Antibody-Dependent-Cellular-Cytotoxicity (ADCC). The pharmaceutical composition according to any one of claims 9 to 11, wherein at least a part of the antibody binds to NK cells.